The present invention relates to novel enzyme preparations comprising an enzyme exhibiting endoglucanase activity which performs very good in industrial applications such as laundry compositions, for biopolishing of newly manufactured textiles, for providing an abraded look of cellulosic fabric or garment, and for treatment of paper pulp. Further, the invention relates to DNA constructs encoding such enzymes, a method for providing a gene encoding for such enzymes, a method of producing the enzymes, enzyme preparations containing such enzymes, and the use of these enzymes for a number of industrial applications.
Cellulases or cellulloytic enzymes are enzymes involved in hydrolyses of cellulose. In the hydrolysis of native cellulose, it is known that there are three major types of cellulase enzymes involved, namely cellobiohydrolase (1,4-xcex2-D-glucan cellobiohydrolase, EC 3.2.1.91), endo-xcex2-1,4-glucanase (endo-1,4-xcex2-D-glucan 4-glucanohydrolase, EC 3.2.1.4) and xcex2-glucosidase (EC 3.2.1.21).
Cellulases are synthesized by a large number of microorganisms which include fungi, actinomycetes, myxobacteria and true bacteria but also by plants. Especially endoglucanases of a wide variety of specificities have been identified.
A very important industrial use of cellulytic enzymes is the use for treatment of cellulosic textile or fabric, e.g. as ingredients in detergent compositions or fabric softener compositions, for bio-polishing of new fabric (garment finishing), and for obtaining a xe2x80x9cstone-washedxe2x80x9d look of cellulose-containing fabric, especially denim, and several methods for such treatment have been suggested, e.g. in GB-A-1 368 599, EP-A-0 307 564 and EP-A-0 435 876, WO 91/17243, WO 91/10732, WO 91/17244, PCT/DK95/000108 and PCT/DK95/00132.
Another important industrial use of cellulytic enzymes is the use for treatment of paper pulp, e.g. for improving the drainage or for deinking of recycled paper.
Especially the endoglucanases (EC No. 3.2.1.4) constitute an interesting group of hydrolases for the mentioned industrial uses. Endoglucanases catalyses endo hydrolysis of 1,4-xcex2-D-glycosidic linkages in cellulose, cellulose derivatives (such as carboxy methyl cellulose and hydroxy ethyl cellulose), lichenin, xcex2-1,4 bonds in mixed xcex2-1,3 glucans such as cereal xcex2-D-glucans or xyloglucans and other plant material containing cellulosic parts. The authorized name is endo-1,4-xcex2-D-glucan 4-glucano hydrolase, but the abbreviated term endoglucanase is used in the present specification. Reference can be made to T.-M. Enveri, xe2x80x9cMicrobial Cellulasesxe2x80x9d in W. M. Fogarty, Microbial Enzymes and Biotechnology, Applied Science Publishers, p. 183-224 (1983); Methods in Enzymology, (1988) Vol. 160, p. 200-391 (edited by Wood, W. A. and Kellogg, S. T.); Bxc3xa9guin, P., xe2x80x9cMolecular Biology of Cellulose Degradationxe2x80x9d, Annu. Rev. Microbiol. (1990), Vol. 44, pp. 219-248; Bxc3xa9guin, P. and Aubert, J-P., xe2x80x9cThe biological degradation of cellulosexe2x80x9d, FEMS Microbiology Reviews 13 (1994) p.25-58; Henrissat, B., xe2x80x9cCellulases and their interaction with cellulosexe2x80x9d, Cellulose (1994), Vol. 1, pp. 169-196.
Fungal endoglucanases have been described in numerous publications, especially those derived from species as e.g. Fusarium oxysporum, Trichoderma reesei, Trichoderma longibrachiatum, Aspergillus aculeatus, Neocallimastix patriciarum, and e.g. from species of the genera Piromyces, Humicola, Myceliophthora, Geotricum, Penicillium, Irpex, Coprinus.
For example, fungal endoglucanases have been described by Sheppard, P. O., et al., xe2x80x9cThe use of conserved cellulase family-specific sequences to clone Cellulase homologue cDNAs from Fusarium oxysporum, Gene, (1994), Vol. 15, pp. 163-167, Saloheimo, A., et al., xe2x80x9cA novel, small endoglucanase gene, egI5, from Trichoderma reesei isolated by expression in yeastxe2x80x9d, Molecular Microbiology (1994), Vol. 13(2), pp. 219-228; van Arsdell, J. N. et al., (1987), Cloning, characterization, and expression in Saccharomyces cerevisiae of endoglucanase I from Trichoderma reesei, Bio/Technology 5: 60-64; Penttilxc3xa4, M. et al., (1986), xe2x80x9cHomology between cellulase genes of Trichoderma reesei: complete nucleotide sequence of the endoglucanase I genexe2x80x9d, Gene 45:253-263; Saloheimo, M. et al, (1988), xe2x80x9cEGIII, a new endoglucanase from Trichoderma reesei: the characterization of both gene and enzymexe2x80x9d, Gene 63:11-21; Gonzxc3xa1les, R., et al., xe2x80x9cCloning, sequence analysis and yeast expression of the egl1 gene from Trichoderma longibrachiatumxe2x80x9d, Appl. Microbiol. Biotechnol., (1992), Vol. 38, pp. 370-375; Ooi, T. et al. xe2x80x9cCloning and sequence analysis of a cDNA for cellulase (FI-CMCase) from Aspergillus aculeatusxe2x80x9d, Curr. Genet., (1990), Vol. 18, pp. 217-222; Ooi, T. et al, xe2x80x9cComplete nucleotide sequence of a gene coding for Aspergillus aculeatus cellulase (FI-CMCase)xe2x80x9d, Nucleic Acids Research, (1990), Vol. 18, No. 19, p. 5884; Xue, G. et al., xe2x80x9cCloning and expression of multiple cellulase cDNAs from the anaerobic rumen fungus Neocallimastix patriciarum in E. colixe2x80x9d, J. Gen. Microbiol., (1992), Vol. 138, pp. 1413-1420; Xue, G. et al., xe2x80x9cA novel polysaccharide hydrolase cDNA (celD) from Neocallimastix patriciarum encoding three multi-functional catalytical domains with high endoglucanase, cellobiohydrolase and xylanase activitiesxe2x80x9d, J. Gen. Microbiol., (1992), Vol. 138, pp. 2397-2403; Zhou, L. et al., xe2x80x9cIntronless celB from the anaerobic fungus Neocallimastix patriciarum encodes a modular family A endoglucanasexe2x80x9d, Biochem. J., (1994), Vol. 297, pp. 359-364; Dalbxc3x8ge, H. and Heldt-Hansen, H. P., xe2x80x9cA novel method for efficient expression cloning of fungal enzyme genesxe2x80x9d, Mol. Gen. Genet., (1994), Vol. 243, pp. 253-260; Ali, B. R. S. et al., xe2x80x9cCellulases and hemicellulases of the anaerobic fungus Piromyces constitute a multiprotein cellulose-binding complex and are encoded by multigene familiesxe2x80x9d, FEMS Microbiol. Lett., (1995), Vol. 125, No. 1, pp. 15-21. Further, the DNA Data Bank of Japan (DDBJ database publicly available at Internet) comprises two DNA sequences cloned from Penicillium janthinellum encoding endoglucanases (cloned by A. Koch and G. Mernitz, respectively) and a DNA sequence cloned from Humicola grisea var. thermoidea encoding an endoglucanase (cloned by T. Uozumi). Two endoglucanases from Macrophomina phaseolina have been cloned and sequenced, see Wang, H. Y. and Jones, R. W.: xe2x80x9cCloning, characterization and functional expression of an endoglucanase-encoding gene from the phytopathogenic fungus Macrophomina phaseolinaxe2x80x9d in Gene, 158:125-128, 1995, and Wang, H. Y. and Jones, R. W.: xe2x80x9cA unique endoglucanase-encoding gene cloned from the phytopathogenic fungus Macrophomina phaseolinaxe2x80x9d in Applied And Environmental Microbiology, 61:2004-2006, 1995. One of these endoglucanases shows high homology to the egl3 endoglucanase from the fungus Trichoderma reesei, the other shows homology to the egl1 from the microbial phytopathogen Pseudomonas solanacearum indicating that both endoglucanases belong to family 5 of glycosyl hydrolases (B. Henrissat, Biochem J 280:309-316 (1991)). Filament-specific expression of a cellulase gene in the dimorphic fungus Ustilago maydis is disclosed in Schauwecker, F. et al. (1995).
WO 91/17243 (Novo Nordisk A/S) discloses a cellulase preparation consisting of a homogenous endoglucanase component immunoreactive with an antibody raised against a highly purified 43 kDa endoglucanase derived from Humicola insolens, DSM 1800; WO 91/17244 (Novo Nordisk A/S) discloses a new (hemi)cellulose degrading enzyme, such as an endoglucanase, a cellobiohydrolase or a xcex2-glucosidase, which may be derived from fungi other than Trichoderma and Phanerochaete; WO 93/20193 discloses an endoglucanase derivable from Aspergillus aculeatus; WO 94/21801 (Genencor Inc.) concerns a cellulase system isolated from Trichoderma longibrachiatum exhibiting endoglucanase activity; WO 94/26880 (Gist Brocades N.V.) discloses an isolated mixture of cellulose degrading enzymes, which preferable are obtained from Trichoderma, Aspergillus or Disporotrichum, comprising endoglucanase, cellobiohydrolase, and xyloglucanase activity; and WO 95/02043 (Novo Nordisk A/S) describes an enzyme with endoglucanase activity derived from Trichoderma harzianum, which can be used for a number of purposes including e.g. degradation or modification of plant cell walls.
It is also known that cellulases may or may not have a cellulose binding domain (a CBD). The CBD enhances the binding of the enzyme to a cellulose-containing fiber and increases the efficacy of the catalytic active part of the enzyme.
There is an ever existing need for providing novel cellulase enzyme preparations which may be used for applications where cellulase, preferably an endoglucanase, activity is desirable.
The object of the present invention is to provide novel enzyme preparations having substantial cellulytic activity at acid, neutral or alkaline conditions and improved performance in paper pulp processing, textile treatment, laundry processes or in animal feed; preferably novel cellulases, more preferably well-performing endoglucanases, which are contemplated to be producible or produced by recombinant techniques.
Surprisingly, it has been found that a group of endoglucanases having certain unique characteristics perform very good in those industrial applications for which endoglucanases are conventionally used. These unique characteristics can be described in terms of conserved regions of the amino acid sequence of the enzyme protein and the inventors have found that cellulytic enzymes, i.e. enzymes exhibiting cellulytic activity, having certain conserved regions are very effective e.g. in the treatment of laundry, in the treatment of newly manufactured textile, in the treatment of papermaking pulp.
Accordingly, in its first aspect the present invention relates to an enzyme preparation consisting essentially of an enzyme having cellulytic activity and comprising a first amino acid sequence consisting of 14 amino acid residues having the following sequence
and a second amino acid sequence consisting of 5 amino acid residues having the following sequence
wherein,
in position 3 of the first sequence, the amino acid is Trp, Tyr or Phe;
in position 4 of the first sequence, the amino acid is Trp, Tyr or Phe;
in position 8 of the first sequence, the amino acid is Arg, Lys or His;
in position 9, 10, 12 and 14, respectively, of the first sequence, and in position 4 of the second sequence, the amino acid is any of the 20 naturally occurring amino acid residues with the provisos that, in the first amino acid sequence, (i) when the amino residue in position 12 is Ser, then the amino acid residue in position 14 is not Ser, and (ii) when the amino residue in position 12 is Gly, then the amino acid residue in position 14 is not Ala.
This surprising finding of clearly recognisable conserved regions, in spite of rather prominent variations found within well-performing endoglucanase enzymes, is a result of studies of a number of fungal DNA sequences encoding for specific amino acid sequences of enzymes having significant cellulytic, especially endoglucanase, activities.
Based on this finding, a novel molecular method taylored to screen specifically for genomic DNA or cDNA characterised by encoding the enzymes of the invention has been developed. As tools for this three sets of degenerated primers were constructed. Accordingly, in its second aspect, the invention relates to a method for providing a gene encoding for cellulytic enzymes having the above conserved regions.
By using this method, i.e. the set of primers for a PCR screening on genomic DNA, it was surprisingly found that DNA encoding for said enzymes can be found from a broad range of fungi, belonging to taxonomically very different organisms and inhabiting ecologically very different niches.
Further, by using this method it has been possible to find DNA sequences encoding for the core regions (catalytically active regions or domains) of said enzymes without any attached cellulose binding domain (CBD) which core regions of enzymes would not have been selected by using conventional performance based screening approaches. The inventors have verified experimentally that the linking of a CBD region to a core region enzyme (comprising the catalytically active region or domain of the enzyme) of the present invention results in a significantly improved performance, e.g. a fifty times higher performance, of the multiple domain enzyme.
Accordingly, the present invention provides novel cellulases, especially endoglucanases, having improved performance in industial applications, either in their native form, or homo- or heterologously produced.
In further aspects, the present invention relates to novel cellulytic enzyme preparations which are derivable from taxonomically specific phyli, classes, orders, families, genera, and species; e.g. from Basidiomycotous Hymenomycetes, Zygomycota, Chytridiomycota; or from the classes Discomycetes, Loculoascomycetes, Plectomycetes; Archaeascomycetes, Hemiascomycetes or from the orders Diaportales, Xylariales, Trichosphaeriales, Phyllachorales; or from the families Nectriaeae, Sordariaceae, Chaetomiaceae, Ceratostomaceae, Lasiosphaeriaceae; or from the genera Cylindrocarpon, Gliocladium, Volutella, Scytalidium, Acremonium, or from the species Fusarium lycopersici, Fusarium passiflora, Fusarium solani, Fusarium anguioides, Fusarium poae, Humicola nigrescens, Humicola grisea, especially such consisting essentially of an enzyme comprising an amino acid sequence selected from the group consisting of the sequences (SEQ ID NOS:105-107)
wherein, in position 4, Xaa is Trp, Tyr or Phe; and in position 1 and 7, Xaa is any of the 20 naturally occurring amino acid residues.
More specifically, the enzyme preparation of the invention is preferably obtainable from the taxonomically specific phyli, classes, orders, families, genera, and species mentioned above which all produce endoglucanases comprising a first peptide consisting of 13 amino acid residues having the following sequence (SEQ ID NO:108)
and a second peptide consisting of 5 amino acid residues having the following sequence (SEQ ID NO:80)
wherein, in position 3 of the first sequence, the amino acid is Trp, Tyr or Phe; in position 4 of the first sequence, the amino acid is Trp, Tyr or Phe; in position 8 of the first sequence, the amino acid is Arg, Lys or His; in position 9, 10, and 12, respectively, of the first sequence, and in position 4 of the second sequence, the amino acid is any of the 20 naturally occurring amino acid residues.
In yet further aspects, the present invention provides DNA constructs comprising a DNA sequence encoding an enzyme exhibiting endoglucanase activity, which DNA sequence comprises the DNA sequence shown in SEQ ID No. 1, 7, 9, 11, 13, 15, 21, and 25, respectively, or analogues thereof.
The present invention also relates to a recombinant expression vector comprising a DNA construct of the invention; to a cell comprising a DNA construct or a recombinant expression vector of the invention; to a method of producing an enzyme, e.g. a recombinant enzyme, of the invention; to a method of providing colour clarification of laundry by using the enzyme of the invention; to a laundry composition comprising the enzyme of the invention; to uses of the enzyme of the invention for degradation or modification of plant material, e.g. cell walls, for treatment of fabric, textile or garment, for treatment of paper pulp; and to an enzyme preparation which is enriched in an enzyme of the present invention.